Tiphaine Gaillard

Molecular surveillance of drug-resistant Plasmodium vivax using pvdhfr, pvdhps and pvmdr1 markers in Nouakchott, Mauritania

OBJECTIVES Plasmodium falciparum and Plasmodium vivax occur in Mauritania. Drug-resistant P. falciparum has been reported, but the drug-resistance status of P. vivax is unknown. The aims of the present study were to determine the prevalence of mutant pvdhfr, pvdhps and pvmdr1 genes and of pvmdr1 gene amplification in P. vivax isolates in Nouakchott, the capital city of Mauritania, and to establish a baseline for molecular surveillance of drug-resistant P. vivax in the country. PATIENTS AND METHODS Between 2007 and 2009, 439 febrile patients were screened for malaria in Nouakchott. The sequences of pvdhfr, pvdhps and pvmdr1 markers in 110 P. vivax isolates were determined by direct sequencing of PCR products. The pvmdr1 gene copy number was determined by real-time PCR. RESULTS The majority of the isolates with a successful PCR amplification (76/86, 88%) were characterized to be of the wild-type pvdhfr genotype, while the remaining 10 isolates carried the S58R and S117N double mutations. All isolates had the wild-type pvdhps genotype SAKAV. For pvmdr1, 75 of 103 (73%) had the wild-type Y976, and 28 (27%) carried the mutant F976. Most (98%) carried the mutant L1076 codon. Of 105 isolates, 102 (97%) had one copy and 3 (3%) had two copies of the pvmdr1 gene. CONCLUSIONS The prevalence of mutations associated with antifolate resistance is low in Mauritania. Further studies are required to determine the roles of pvmdr1 mutations and gene amplification in conferring drug resistance. These data will serve as a baseline for further monitoring of drug-resistant malaria.

Multinormal in vitro distribution of Plasmodium falciparum susceptibility to piperaquine and pyronaridine.

BackgroundIn 2002, the World Health Organization recommended that artemisinin-based combination therapy (ACT) be used to treat uncomplicated malaria. Dihydroartemisinin-piperaquine and artesunate-pyronaridine are two of these new combinations. The aim of the present work was to assess the distribution of the in vitro values of pyronaridine (PND) and piperaquine (PPQ) and to define a cut-off for reduced susceptibility for the two anti-malarial drugs.MethodsThe distribution and range of the 50% inhibitory concentration values (IC50) of PND and PPQ were determined for 313 isolates obtained between 2008 and 2012 from patients hospitalized in France for imported malaria. The statistical Bayesian analysis was designed to answer the specific question of whether Plasmodium falciparum has different phenotypes of susceptibility to PND and PPQ.ResultsThe PND IC50 values ranged from 0.6 to 84.6 nM, with a geometric mean of 21.1 ± 16.0 nM (standard deviation). These values were classified into three components. The PPQ IC50 values ranged from 9.8 to 217.3 nM, and the geometric mean was 58.0 ± 34.5 nM. All 313 PPQ values were classified into four components. Isolates with IC50 values greater than 60 nM or four-fold greater than 3D7 IC50 are considered isolates that have reduced susceptibility to PND and those with IC50 values greater than 135 nM or 2.3-fold greater than 3D7 IC50 are considered isolates that have reduced susceptibility to PPQ.ConclusionThe existence of at least three phenotypes for PND and four phenotypes for PPQ was demonstrated. Based on the cut-off values, 18 isolates (5.8%) and 13 isolates (4.2%) demonstrated reduced susceptibility to PND and PPQ, respectively.

Malaria Prophylaxis Failure with Doxycycline, Central African Republic, 2014

To the Editor: Doxycycline is an effective antimalarial prophylactic drug when administered as a monotherapy 1 day before, daily during, and for 4 weeks after travel to an area where malaria is endemic (1). Doxycycline is currently a recommended chemoprophylactic regimen for travelers visiting areas where malaria is endemic and has a high prevalence of chloroquine or multidrug resistance (2). The World Health Organization also recommends doxycycline in combination with quinine or artesunate as the second-line treatment for uncomplicated Plasmodium falciparum malaria (3). Prophylactic and clinical failures of doxycycline against P. falciparum have been associated with both inadequate doses (4) and poor patient compliance (5). However, resistance can also explain failures of prophylaxis. Cycline resistance in Plasmodium spp. has been documented as a consequence of selective drug pressure in a P. berghei murine malaria model (6). The administration of increasing doses of minocycline to mice infected with 1 × 107 parasites for 86 successive passages over 600 days made it possible to obtain a resistant P. berghei strain with a median drug inhibitory concentration (IC50) of 600 mg/kg/d, which is 6-fold higher than that of the susceptible starting strain (100 mg/kg/d) (6). A Bayesian mixture modeling approach identified 3 different phenotypes (low, medium, and high doxycycline IC50 phenotypic groups) among P. falciparum clinical isolates (7,8). Using 90 isolates from 14 countries, we demonstrated that increases in copy numbers of P. falciparum metabolite drug transporter gene (Pfmdt, PFE0825w) and P. falciparum GTPase TetQ gene (PfTetQ, PFL1710c) are associated with reduced susceptibility to doxycycline (9); this association was later confirmed (7). In addition, isolates with PfTetQ KYNNNN motif repeats are associated with in vitro reduced susceptibility to doxycycline and with a significantly higher probability of having an IC50 above the doxycycline resistance threshold of 35 μM (9,10). We report a case of documented malaria prophylactic failure with doxycycline in a 26-year-old soldier from France who was infected during a 6-week peacekeeping mission in the Central African Republic in 2014. According to his colleagues and the collective prophylaxis intake, the patient had been compliant with doxycycline prophylaxis. On admission to a hospital in Bangui, Central African Republic, the patient had fever (temperature 40°C), alteration of consciousness, and hypotension. The diagnosis of severe P. falciparum malaria was made on the basis of a rapid diagnostic test confirmed by a blood smear test (parasitemia 8% on day 0). Intravenous artesunate was immediately started, in accordance with World Health Organization recommendations (3). The patient’s clinical condition worsened, and kidney failure developed. Twenty-four hours later (day 1), he was transported by airplane to Begin Military Teaching Hospital (Saint-Mande, France). On admission, he had cerebral edema and a P. falciparum parasitemia level of 0.7%. The patient died 1 day later (day 2). A blood sample obtained from the patient on day 1 in France showed a doxycycline concentration of 195 μg/mL plasma. This concentration, which was determined by liquid chromatography coupled with tandem mass spectrometry, was compatible with a last doxycycline uptake 1 day before diagnosis (day −1). The finding of the expected doxycycline plasma concentration, together with assurances (colleague’s statements and collective intake of doxycycline) that the patient had followed the drug regimen, was sufficient to suggest prophylaxis failure in a treatment-compliant patient. The P. falciparum sample obtained from the patient on arrival in France was evaluated for in vitro susceptibility to doxycycline, but the evaluation was unsuccessful. The number of copies of PfTetQ and Pfmdt genes were evaluated relative to the single-copy P. falciparum β-tubulin gene (pfβtubulin), as previously described (7,8). The sample was assayed in triplicate. The 2–ΔΔCt method (where Ct indicates cycle threshold) of relative quantification was used and adapted to estimate the number of copies of Pfmdt and PfTetQ by using the formula ΔΔCt = (Ct (PfTetQ or Pfmdt) − Ct (Pfβtubulin))Sample – (Ct (PfTetQ or Pfmdt) − Ct (Pfβtubulin))Calibrator. Genomic DNA extracted from 3D7 P. falciparum, which has a single copy of each gene, was used for calibrator sample; Pfβtubulin served as the control housekeeping gene. The experiment was assayed twice. The sample had 2 copies of PfTetQ and Pfmdt genes, which suggested decreased in vitro susceptibility of the sample to doxycycline (8,9). The genotyping of PfTetQ sequence polymorphisms was done by using conventional methods with the primers PfTetQ forward (5′-TCACGACAAATGTGCTAGATAC-3′) and PfTetQ reverse (5′-ATCATCATTTGTGGTGGATAT-3′), as previously described (10). Two PfTetQ KYNNNN motif repeats were found in the sample; 35 μM (odds ratio 15) (10). The 2 copies of Pfmdt and the 2 KYNNNN motif repeats have been shown to be associated with parasites with in vitro resistance to doxycycline (9,10). The association of doxycycline resistance (prophylactic failure with statement of correct intake and the presence of an expected concentration) with increased Pfmdt copies and decreased PfTetQ KYNNNN motif repeats suggest that these molecular markers are predictive markers of doxycycline resistance that can be used for resistance surveillance.

Antibiotics in malaria therapy: which antibiotics except tetracyclines and macrolides may be used against malaria?

Malaria, a parasite vector-borne disease, is one of the most significant health threats in tropical regions, despite the availability of individual chemoprophylaxis. Malaria chemoprophylaxis and chemotherapy remain a major area of research, and new drug molecules are constantly being developed before drug-resistant parasites strains emerge. The use of anti-malarial drugs is challenged by contra-indications, the level of resistance of Plasmodium falciparum in endemic areas, clinical tolerance and financial cost. New therapeutic approaches are currently needed to fight against this disease. Some antibiotics that have shown potential effects on malaria parasite have been recently studied in vitro or in vivo intensively. Two families, tetracyclines and macrolides and their derivatives have been particularly studied in recent years. However, other less well-known have been tested or are being used for malaria treatment. Some of these belong to older families, such as quinolones, co-trimoxazole or fusidic acid, while others are new drug molecules such as tigecycline. These emerging antibiotics could be used to prevent malaria in the future. In this review, the authors overview the use of antibiotics for malaria treatment.

Macrolides and associated antibiotics based on similar mechanism of action like lincosamides in malaria

Malaria, a parasite vector-borne disease, is one of the biggest health threats in tropical regions, despite the availability of malaria chemoprophylaxis. The emergence and rapid extension of Plasmodium falciparum resistance to various anti-malarial drugs has gradually limited the potential malaria therapeutics available to clinicians. In this context, macrolides and associated antibiotics based on similar mechanism of action like lincosamides constitute an interesting alternative in the treatment of malaria. These molecules, whose action spectrum is similar to that of tetracyclines, are typically administered to children and pregnant women. Recent studies have examined the effects of azithromycin and the lincosamide clindamycin, on isolates from different continents. Azithromycin and clindamycin are effective and well tolerated in the treatment of uncomplicated malaria in combination with quinine. This literature review assesses the roles of macrolides and lincosamides in the prophylaxis and treatment of malaria.

Delayed Onset of Plasmodium falciparum Malaria after Doxycycline Prophylaxis in a Soldier Returning from the Central African Republic

Emilie Javelle, Marylin Madamet, Tiphaine Gaillard, Guillaume Velut, Corinne Surcouf, Rémy Michel, Eric Garnotel, Fabrice Simon, Bruno Pradines Service de Maladies Infectieuses et Tropicales, Hôpital d’Instruction des Armées Laveran, Marseille, France; Aix Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM 63, CNRS 7278, IRD 198, Inserm 1095, Marseille, France; Equipe Résidente de Recherche en Infectiologie Tropicale, Institut de Recherche Biomédicale des Armées, Hôpital d’Instruction des Armées Laveran, Marseille, France; Centre National de Référence du Paludisme, Marseille, France; Fédération des Laboratoires, Hôpital d’Instruction des Armées Saint Anne, Toulon, France; Service de Santé Publique, Centre d’Epidémiologie et de Santé Publique des Armées, Marseille, France; Fédération des Laboratoires, Hôpital d’Instruction des Armées Laveran, Marseille, France; Service de Surveillance Epidémiologique, Centre d’Epidémiologie et de Santé Publique des Armées, Marseille, France; Ecole du Val-de-Grâce, Paris, France; Unité de Parasitologie et d’Entomologie, Département des Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, Brétigny sur Orge, France

Molecular Markers and In Vitro Susceptibility to Doxycycline in Plasmodium falciparum Isolates from Thailand

ABSTRACT Determinations of doxycycline 50% inhibitory concentrations (IC50) for 620 isolates from northwest Thailand were performed via the isotopic method, and the data were analyzed by the Bayesian method and distributed into two populations (mean IC50s of 13.15 μM and 31.60 μM). There was no significant difference between the group with low IC50s versus the group with high IC50s with regard to copy numbers of the Plasmodium falciparum tetQ (pftetQ) gene (P = 0.11) or pfmdt gene (P = 0.87) or the number of PfTetQ KYNNNN repeats (P = 0.72).

Absence of association between Plasmodium falciparum small sub-unit ribosomal RNA gene mutations and in vitro decreased susceptibility to doxycycline

BackgroundDoxycycline is an antibiotic used in combination with quinine or artesunate for malaria treatment or alone for malaria chemoprophylaxis. Recently, one prophylactic failure has been reported, and several studies have highlighted in vitro doxycycline decreased susceptibility in Plasmodium falciparum isolates from different areas. The genetic markers that contribute to detecting and monitoring the susceptibility of P. falciparum to doxycycline, the pfmdt and pftetQ genes, have recently been identified. However, these markers are not sufficient to explain in vitro decreased susceptibility of P. falciparum to doxycycline. In this paper, the association between polymorphism of the small sub-unit ribosomal RNA apicoplastic gene pfssrRNA (PFC10_API0057) and in vitro susceptibilities of P. falciparum isolates to doxycycline were investigated.MethodsDoxycycline IC50 determinations using the hypoxanthine uptake inhibition assay were performed on 178 African and Thai P. falciparum isolates. The polymorphism of pfssrRNA was investigated in these samples by standard PCR followed by sequencing.ResultsNo point mutations were found in pfssrRNA in the Thai or African isolates, regardless of the determined IC50 values.ConclusionsThe pfssrRNA gene is not associated with in vitro decreased susceptibility of P. falciparum to doxycycline. Identifying new in vitro molecular markers associated with reduced susceptibility is needed, to survey the emergence of doxycycline resistance.